1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal.

Justification for type of information:

Data is from peer reviewed journal.

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Biodegradation study of the test chemical was carried out for determining the biodegradability rate of the test substance.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (IUPAC Name): 1-(2-Methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol
- Common name: Sudan IV
- Molecular formula: C24H20N4O
- Molecular weight: 380.449 g/mol
- Smiles notation: c12c(\N=N\c3c(cc(\N=N\c4c(cccc4)C)cc3)C)c(ccc1cccc2)O
- InChI: 1S/C24H20N4O/c1-16-7-3-6-10-21(16)26-25-19-12-13-22(17(2)15-19)27-28-24-20-9-5-4-8-18(20)11-14-23(24)29/h3-15,29H,1-2H3/b26-25+,28-27+
- Substance type: Organic
- Physical state: Solid
- Other: Test chemical was purchased from Sigma Chemical Co.

Oxygen conditions:

anaerobic

Inoculum or test system:

other: Bacteria

Details on inoculum:

- Laboratory culture: The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).

- Method of cultivation: The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 1.5 µg/ml, the cultures were incubated at 37 ᵒC in an anaerobic chamber for 2 days without agitation.

- Storage conditions: All strains were preserved at -80 ᵒC in 10 to 15% glycerol stocks and revived as needed.

- Preparation of inoculum for exposure: The strains, except for Lactobacillus species, which were routinely cultured on deMann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37 ᵒC under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide.

- Pretreatment:: The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture.

Duration of test (contact time):

2 d

Initial conc.:

1.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

(% degradation)

Details on study design:

TEST CONDITIONS
- Composition of medium: Brain Heart Infusion (BHI) broth or deMann-Rogosa-Sharpe (MRS) broth or agar was used.
- Additional substrate: vitamin k, hemin.
- Test temperature: 37 ᵒC
- Other: the cultures were incubated in an anaerobic chamber for 2 days without agitation.

TEST SYSTEM
- Culturing apparatus: Erlenmeyer flask


CONTROL AND BLANK SYSTEM
- Inoculum blank: Two control consisted of sterile liquid medium and sterile liquid medium with bacteria.
- Abiotic sterile control: one abiotic control was setup using sterile liquid medium with dyes.

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

2 d

Remarks on result:

other: Other details not known

Details on results:

Test substance undergoes 100% degradation by bacterial strains such as B. vulgatus, B.ovatus, B. uniformis, B. distasonis, B. fragilis, B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. indolis, C. clostridioforme , E. aerofaciens, E. limosum , E. faecalis, E. faecium, F. russi, F. nucleatum , L. paracasei , L. reuteri, L. rhamnosus, R. obeum and R. gnavus, respectively.
The bacteria which are unable to degrade the test chemical are Bifidobacterium catenulatum, Clostridium ramosum, Eubacterium tenue, Escherichia coli and Peptostreptococcus magnus.

Table: Test chemical reduction by thirty five prevalent human intestinal bacterial species.

 

Human intestinal bacterial species	Biodegradation (%)
Bacteroides vulgatusATCC 8482	100
Bacteroides ovatusATCC 8483	100
Bacteroides uniformisATCC	100
Bacteroides distasonisATCC 8503	100
Bacteroides fragilisATCC 23745	100
Bacteroides thetaiotaomicronATCC 29148	100
Bacteroides caccaeATCC 43185	100
Bifidobacterium longumATCC 15707	60
Bifidobacterium adolesecentisATCC 15703	40
Bifidobacterium infantisATCC 15697	100
Bifidobacterium catenulatumATCC 27539	-
Bifidobacterium angulatumATCC 27535	40
Clostridium perfringensATCC 13124	100
Clostridium butyricumATCC 19398	60
Clostridium ramosumATCC 25582	-
Clostridium difficle ATCC 9689	60
Clostridium indolisATCC 25771	100
Clostridium leptumATCC 29065	60
Clostridium clostridioformeATCC 29084	100
Eubacterium aerofaciensATCC 25986	100
Eubacterium limosumATCC 8486	100
Eubacterium tenueATCC 25553	-
Enterococcus faecalisATCC 27274	100
Enterococcus faeciumATCC 19434	100
Escherichia coliATCC 25922	-
Fusobacterium russiATCC 25533	100
Fusobacterium nucleatumATCC 25586	100
Lactobacillus bifidus ATCC 11146	80
Lactobacillus paracaseiATCC 27092	100
Lactobacillus reuteriATCC 23272	100
Lactobacillus rhamnosusATCC 53103	100
Lactobacillus ruminisATCC 25644	40
Peptostreptococcus magnusATCC 14955	-
Ruminococcus obeumATCC 29174	100
Ruminococcus gnavusATCC 29149	100

 

 

The metabolite produced from test chemical by E. faecalis was identified as o-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH⁺+acetonitrile]. 2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical was not degraded by the bacterium.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Among the tested bacterial strains, B. vulgatus, B.ovatus, B. uniformis, B. distasonis, B. fragilis , B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. indolis, C. clostridioforme , E. aerofaciens, E. limosum , E. faecalis, E. faecium, F. russi, F. nucleatum , L. paracasei , L. reuteri, L. rhamnosus, R. obeum and R. gnavus were able to completely degrade (100%) the test chemical. The metabolite produced from test chemical by E. faecalis was identified as o-toluidine, based on an identical retention time of 7.42 min and ions at m/z 108 [MH+] and 149 [MH+ + acetonitrile]. 2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical was not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

Executive summary:

Biodegradation study of test chemical was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days.The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various test chemical conc.. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Test chemical stock solutions were added to the medium at final concentrations of 1.5µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometry(LC/ESI-MS). Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B. vulgatus, B.ovatus, B. uniformis, B. distasonis, B. fragilis , B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. indolis, C. clostridioforme, E. aerofaciens, E. limosum , E. faecalis, E. faecium, F. russi, F. nucleatum, L. paracasei, L. reuteri, L. rhamnosus, R. obeum and R. gnavus were able to completely degrade (100%) the test chemical.The bacteria which are unable to degrade the test chemical are Bifidobacterium catenulatum, Clostridium ramosum, Eubacterium tenue, Escherichia coli and Peptostreptococcus magnus , respectively. The metabolite produced from test chemical by E. faecalis was identified aso-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH⁺+acetonitrile].2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

Description of key information

Biodegradation study of test chemical was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days (Haiyan Xu et. al., 2010).The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various test chemical conc.. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Test chemical stock solutions were added to the medium at final concentrations of 1.5µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometry(LC/ESI-MS). Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains,B. vulgatus,B.ovatus,B. uniformis, B. distasonis,B. fragilis ,B. thetaiotaomicron,B. caccae,B. infantis, C. perfringens, C. indolis, C. clostridioforme,E. aerofaciens, E. limosum , E. faecalis, E. faecium,F. russi, F. nucleatum,L. paracasei,L. reuteri, L. rhamnosus,R. obeum and R. gnavuswere able to completely degrade (100%) the test chemical.The bacteria which are unable to degrade the test chemical areBifidobacterium catenulatum,Clostridium ramosum, Eubacterium tenue, Escherichia coliandPeptostreptococcus magnus ,respectively. The metabolite produced from test chemical by E. faecalis was identified aso-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH⁺+acetonitrile].2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced byE. coliwas detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Various experimental studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Haiyan Xu et. al., 2010),biodegradation experiment of test chemical was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days.The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various test chemical conc.. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Test chemical stock solutions were added to the medium at final concentrations of 1.5µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometry(LC/ESI-MS). Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B. vulgatus, B.ovatus, B. uniformis, B. distasonis, B. fragilis , B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. indolis, C. clostridioforme, E. aerofaciens, E. limosum , E. faecalis, E. faecium, F. russi, F. nucleatum, L. paracasei, L. reuteri, L. rhamnosus, R. obeum and R. gnavus were able to completely degrade (100%) the test chemical.The bacteria which are unable to degrade the test chemical are Bifidobacterium catenulatum, Clostridium ramosum, Eubacterium tenue, Escherichia coli and Peptostreptococcus magnus , respectively. The metabolite produced from test chemical by E. faecalis was identified aso-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH++acetonitrile].2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

For the test chemical, biodegradation study was performed. The test is carried at a temperature of 37ᵒC with a duration period of 2 days (peer reviewed journal, 2010). The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10 mg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC and Liquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS).Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum were able to completely degrade (100%) the test chemical whereas partial degradation of the test chemical occurred by the bacterial strains such as B. ovatus, B. distasonis, B. thetaiotaomicron, B.caccae, C. clostridioforme, E. faecium, F. russi and L. paracasei. Thus, the test chemical is readily biodegradable in water. The bacteria which are unable to degrade the test chemical areB. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnus and R. gnavus. The metabolite produced from test chemical by E. faecalis was identified as 2,4-dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z 122 [MH+] and 163 [MH+ + acetonitrile]. 1-Amino-2-naphthol from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, the test chemical is considered to be readily biodegradable in water.

 

In a supporting study from peer reviewed journal (D. Brown et. al., 1987),biodegradation experiment was carried out for determining the biodegradability rate of the test chemical. Activated sludge was used as an inoculum and the study was performed under anaerobic conditions at a temperature of 35°C for a period of 56 days. Samples of the aqueous phase were analyzed either qualitatively or quantitatively by an appropriate chromatographic method for the presence of certain of the expected aromatic amine metabolites. The percentage degradation of test chemical was determined to be 100% degradation by appropriate chromatography method in 7 days. The metabolites identified by the appropriate chromatographic method were 4,4 '-diamino-3,3'-dimethyl biphenyl and 4-methyl benzenesulphonic acid- (4'-aminophenyl) ester, respectively. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

On the basis of above results for test chemical, it can be concluded that the test chemical can be considered to be readily biodegradable in nature.